Non-Signal Imidazole Reagents for Mass Spectrometry Analysis of Phosphomonoesters

ABSTRACT

Analytical chemical reagents termed non-signal imidazoles and a method for their use that provide a host of advantages for analysis of phosphomonoesters are described. The method and compounds of the invention provide a host of advantages for the analysis of phosphomonoester-containing compounds, namely characteristic, multi-analyte detection with high sensitivity and specificity of known and unknown phosphomonoester-containing compounds simultaneously.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/692,704, filed Jun. 21, 2005, entitled“ULTRASENSITIVE MASS DETECTION OF ORGANOPHOSPHATES,” the entiredisclosure of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND OF THE INVENTION

Phosphomonoester-containing compounds are an important group to be ableto analyze with accuracy and sensitivity because many bioactivemolecules fall into this class. These molecules include phosphomonoesterforms of nucleosides (e.g., ribonucleotides, deoxyribonucleotides,dideoxyribonucleotides), nucleoside di- and tri-phosphates,deoxynucleoside di- and tri-phosphates, dinucleotides, trinucleotides,oligonucleotides, lipids, oligosaccharides, amino acids, sugars,peptides, metabolites and drugs. High performance analysis is requiredbecause of the multiplicity, diversity, low concentrations, andadsorptive nature of many of these phosphomonoesters, making theiranalysis difficult.

Aryl groups such as phenyl, pyridyl and naphthyl are common parts oforganic compounds. Often an aryl group in a compound is substituted witha group such as bromo, chloro, alkyl (such as methyl), alkoxy (such asmethoxy), deutero or amide. This substitution modifies the chemical orphysical properties of the compound to one degree or another. More thanone substitutent may be present on an aryl group.

Mass spectrometry is an important technique for analyzing many chemicalsubstances. At its best, it provides characteristic multi-analytedetection with high sensitivity and specificity. However, rarely arethese advantages brought together in a single method. In general, thesensitivity of mass spectrometry is analyte and sample dependent, andvaries with the conditions in the mass spectrometer. Even whenconditions are optimized for each analyte of interest, and each analyteis detected under the best possible conditions, responses can varywidely for different analytes. Different analytes also tend to fragmentto different degrees in the mass spectrometer. The most intense fragmentmay come from only a small part of the overall structure of a compound,providing little structural characterization. This failure of massspectrometry to achieve its full analysis potential exists for all formsof mass spectrometry. This includes the common techniques of matrixassisted laser desorption ionization mass spectrometry (MALDI-MS), andelectrospray ionization mass spectrometry (ESI-MS), including MS/MSforms of these techniques. Unfortunately, phosphomonoester compounds areamong the worst in their present capability of being analyzed with highperformance by mass spectrometry.

Stable isotope reagents, in which a chemical reagent is enriched in astable isotope such as deuterium, are widely used in mass spectrometry.In one use, a known amount of a stable isotope form of the analyte isadded to a sample to provide a more accurate analysis of this samplebased on the principle of isotope dilution. In another use,corresponding covalent labeling reagents as an isotope duo (one ordinaryand one isotope-enriched) are used separately so that the target analyteis labeled in one sample with an ordinary reagent, and the same analytein the second sample is labeled with the corresponding stable isotopereagent. The samples then can be combined prior to subsequent cleanupsteps before analysis by mass spectrometry, revealing the relativeamount of the target analyte in the two samples. In a third use, a givensample is reacted with a combined isotopic duo in order to convert ananalyte into a pair of products that give a mass-distinctive, splitsignal in the mass spectrometer to enhance specificity. Stable isotopeforms of test substances are also used in mass spectrometry to sort outfragmentation pathways and identify exchangeable atoms.

The sensitivity for detection of phosphomonoester compounds can beincreased and made relatively uniform by labeling the compounds with apre-existing signal group such as a fluorescent dye or radioisotope thatinherently provides intensive detection properties by the correspondingdetection technique for the signal group employed. For example, Gieseand Wang (U.S. Pat. No. 5,512,486) introduced imidazole reagentscontaining pre-existing signal groups for labeling of phosphomonoestercompounds to improve detection sensitivity by the designated detectiontechnique for the pre-existing signal group. One of these reagents,containing a pre-existing fluorescent signal group, was used to converta nucleotide into a corresponding fluorescent phosphorimidazolide, andthe latter compound then was detected by fluorescence detection. Thestructure of the compound was confirmed by MALDI-TOF-MS. However, highsensitivity was not demonstrated by this MS technique, since thesmallest amount of fluorescent phosphorimidazolide detected in theinstrument was 30 picomoles. High specificity was not demonstrated sincethere was no isotopic duo. Multi-analyte detection was not encouraged,since several fragmentation peaks were formed by the fluorescentphosphorimidazolide product. There was no evidence that differentnucleotides could give a similar response under a single set of MSconditions.

Creation of a signal group by combining an analyte with a non-signalchemical derivatization reagent is known in the field of detection byfluorescence. For example, fluorescamine and o-phthalaldehyde non-signalreagents can be reacted with amine-bearing compounds to form fluorescentproducts.

Characteristic, multi-analyte analysis with high sensitivity andspecificity is important for phosphomonoesters because of the greatnumber and diversity of bioactive compounds in this class. It isimportant to simultaneously detect known and unknown phosphomonoesters,since not all bioactive phosphomonoester compounds may have beendiscovered or identified, and their role needs to be sorted out bothindependently and relative to known phosphomonoesters. High sensitivityis critical especially for human samples which are often limited inamount, and further may have a low concentration of phosphomonoesters.High specificity is important in the analysis of phosphomonoesters toavoid false positive and false negative results.

BRIEF SUMMARY OF THE INVENTION

This invention is directed to analytical methodology, reagents andproducts that provide characteristic, multi-analyte analysis ofphosphomonoester-containing compounds with high sensitivity andspecificity by mass spectrometry. A signal group capable of furnishingthis set of detection properties is created by covalently labeling thephosphomonoester group of the compound to be analyzed with a non-signalimidazole reagent to form an anion signal.

In one aspect, the invention features a class of non-signal imidazolereagents having the general structure of:

wherein either R₁ or R₂ is hydrogen (H) or deuterium (D), B comprisesone to eight carbon atoms, A comprises an aryl group, and at least oneof A or B is substituted with one or more atoms or groups other than Hwhen A is phenyl and B comprises an amide group. In a preferredembodiment, B is substituted with four deuterium atoms and A issubstituted with a bromine atom (Br), a chlorine atom (Cl), or one ormore deuterium atoms. In other embodiments, A comprises a naphthyl orbiphenyl group, or A is substituted with one or more fluorine atoms,alkyl groups or alkoxy groups; B comprises an amide group or an ethergroup. More preferably, B is —CD₂CD₂- and A is R₃C₆H₄CONH—, wherein R₃is Br or Cl.

One form of the invention features an isotopic duo non-signal imidazolereagent that ultimately yields two intense peaks for a signalphosphorimidazolide product. The isotopic duo reagent also can be usedto compare the relative amounts of a phosphomonoester compound in twosamples. The isotopic duo detection option can be practiced, forexample, with natural bromoaryl non-signal imidazole reagents due to thehigh natural abundance of two stable isotopes of bromine. The same istrue for corresponding chloroaryl imidazole reagents. The two-compoundform of an isotopic duo non-signal imidazole reagent can consist of amixture of corresponding enriched and non-enriched non-signal imidazolecompounds in terms of enrichment with a stable isotope such asdeuterium, in order to practice this invention.

In another aspect, the invention features a method for detectingphosphomonoester-containing compounds that includes the steps ofproviding a non-signal imidazole reagent, linking the reagent to thephosphomonoester compounds to form products that each provide aphosphorimidazolide anion signal group, and detecting these products asphosphorimidazolide anions by mass spectrometry. The reagent can be amixture of two or more different non-signal imidazole reagents. Thephosphomonoester-containing compound analyzed is, e.g., a nucleotide,dinucleotide or trinucleotide. Alternatively, thephosphomonoester-containing compound can be, e.g., a phosphopeptide.Preferably, the non-signal imidazole reagent contains an aryl group, andone of the adjacent carbon atoms of the imidazole group is substitutedwith a carbon atom. More preferably, the non-signal imidazole reagent isselected from the group consisting ofN-[2-(1H-imidazol-4-yl)ethyl]benzamide,N-[2-(1H-imidazol-4-yl)ethyl-d₄]benzamide,4-chloro-N-[2-(1H-imidazol-4-yl)ethyl]benzamide,4-bromo-N-[2-(1H-imidazol-4-yl)ethyl]benzamide, andN-[2-(1H-imidazol-4-yl)ethyl]benz-d₅-amide. In the method, the productformed can be detected, e.g., by matrix-assisted laser desorptionionization mass spectrometry.

In a third aspect, the invention features aryl phosphorimidazolideshaving the general structure:

wherein R₁ comprises an aryl group and R₂ is an organic molecule.Preferably, R₁ comprises a phenyl group.

The method and compounds of the invention provide a host of advantagesfor the analysis of phosphomonoester-containing compounds, namelycharacteristic, multi-analyte detection with high sensitivity andspecificity of known and unknown phosphomonoester-compoundssimultaneously. Never before has it been possible to bring theseadvantages together in a single method for the analysis ofphosphomonoester compounds.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings in which:

FIG. 1 shows exemplary non-signal imidazole reagents useful in themethod of the invention;

FIG. 2 shows detection of deoxynucleotides labeled with a non-signalimidazole reagent according to the method of the invention;

FIG. 3 shows detection of 5 fmol of a deoxynucleotide with the method ofthe invention;

FIG. 4 shows the detection, with the method of the invention of anacetylaminofluorene DNA adduct spiked into DNA;

FIG. 5 shows the detection, with the method of the invention, of thefour common deoxynucleotides of DNA, dimethyldeoxyadenosinemonophosphate (tentatively identified), and unknown phosphomonoesterswhen the method was applied to DNA from human placenta;

FIG. 6 shows the detection of ribonucleotides from transfer RNA usingthe method of the invention;

FIG. 7 shows the detection of a phosphopeptide using the method of theinvention;

FIG. 8 shows detection at the amol level using the method of theinvention; and

FIG. 9 shows direct evidence of N⁶ MedA in mammalian DNA.

DETAILED DESCRIPTION OF THE INVENTION

The method and compounds of the invention provide a host of advantagesfor the analysis of phosphomonoester-containing compounds, namelycharacteristic, multi-analyte detection with high sensitivity andspecificity of known and unknown phosphomonoester-compoundssimultaneously. The advantage of characteristic detection refers to theintact, complete molecular mass that the method of the invention canfurnish for a phosphomonoester. The phosphorimidazolide anion signalgroup that is created in the linking step of this method enables theentire product from a phosphomonoester-containing compound to bedetected as a molecular anion, thereby providing a characteristicdetection. Because this invention can detect such products in this waywith similar efficiency under a single set of conditions in the massspectrometer, it also provides the advantage of multi-analyte detectionfor both known and unknown phosphomonoesters.

The advantage of high sensitivity is achieved by this invention througha combination of three critical properties. The first is that an intensesignal group is created in the linking step between thephosphomonoester-containing compound and the non-signal imidazolereagent. When, instead, a pre-existing signal derivatization reagent isemployed in a method, residual signal reagent, signal contaminants inthis reagent, and reaction products of the signal reagent withnonanalyte compounds all can contribute to residual chemical noise atthe end of the derivatization reaction. This reduces sensitivity,keeping in mind that sensitivity is signal over noise. It is veryimportant for high sensitivity that a signal group is created as in themethod of the invention rather than being pre-existing.

The second way in which this invention provides high sensitivity is thatthe phosphorimidazolide anion signal group is created with reagents thatbear a positive charge or acquire a positive charge at low pH. Thisenables efficient removal of residual reagents at the end of thereaction in a simple way, such as by cation exchange chromatography. Thethird property of the invention that contributes to high sensitivity isthat the phosphorimidazolide anion signal product that forms is bothdesorption-prone and fragmentation-resistant in the mass spectrometer.Often the phosphomonoester moiety is labile in the mass spectrometer,but this method stabilizes it. This leads to an intense peak for themolecular phosphorimidazolide anion of the product. The practice of themethod of the invention with an aryl non-signal imidazole reagent andwith MALDI-MS, such as MALDI-TOF-MS or MALDI-TOF/TOF-MS, is preferredfor achieving high sensitivity based on the above three mechanisms forhigh sensitivity.

In addition to the fact that mass spectrometry is well-known as aspecific technique in general, and that phosphorimidazolides can beformed specifically, additional specificity is achieved by thisinvention in two ways, both of which derive from the opportunity toemploy a non-signal imidazole reagent as an isotopic duo. In the firsttechnique, the relative amount of phosphomonoester-containing compoundscan be compared in two samples by subjecting each sample toderivatization with only one member of the isotopic duo reagent. Thesamples then are combined for co-detection by mass spectrometry, wherethe relative peak heights for nonisotopic and isotopic forms of eachphosphomonoester-containing compound provide the relative amounts ofthese compounds in each sample.

In the second strategy for boosting specificity, a given sample islabeled with a combined isotopic duo reagent, and peaks for thephosphomonoester-containing compounds can be discriminated against noisepeaks, when the latter peaks are present in the mass spectrum, based onthe isotopic mass spacing and relative peak heights for the pair ofpeaks from each derivatized phosphomonoester.

Phosphomonoester-containing compounds are at the heart of muchbiochemistry and physiology in both health and disease. The host ofanalytical advantages of this invention enables it to fill in gaps inunderstanding about the role of phosphomonoesters as bioactive agents,and in the ability of investigators to measure phosphomonoesters withhigh performance as biomarkers of health and disease. One example ismetabolomics, the study of biological systems based on profiling many ofthe metabolites simultaneously. Generally, the initial goal is todiscover the metabolic pathways that are disrupted in disease. Knowingthese disruptions helps to find drugs that restore these pathways tonormal and thereby treat the disease. This knowledge can also lead todiscovery of metabolite analytes for clinical diagnostics. Thisinvention enables phosphomonoester metabolites to be profiled.

A second example of an important application of this invention is thestudy of DNA damage by chemicals and radiation. Damage in this way toDNA is termed “DNA adducts.” For more than, 30 years, cancerepidemiologists have wanted a good test for DNA adducts in people as away to help individualize cancer prevention because DNA adductscontribute to the initiation of cancer. The concept is similar to themeasurement of cholesterol to help individualize prevention of heartattacks. Unfortunately, in spite of thousands of research articles onDNA adducts, the trace amounts of DNA adducts in human samples (amillion to a billion times less than level of cholesterol, requiringvery sensitive detection techniques); the unknown structures of most ofthese adducts; the lack of a detection technique for discovering unknownDNA adducts in a specific way; and the limited ability of currentmethods to achieve multi-analyte detection with a relatively uniformresponse has stymied efforts to make the measurement of DNA adducts inhumans a weapon against cancer. The method of the invention overcomesthis barrier by providing the combination of key analytical propertiesthat has been missing. The measurement of DNA adducts in people also isof interest because DNA adducts may contribute to aging, heart disease,infertility, and diabetes.

A third example of an important application of this invention is themeasurement of phosphopeptides. The function of many proteins iscontrolled by phosphorylation. This takes place on the serine, theronineand tyrosine sites of proteins. Disruption of this phosphorylation takesplace in many diseases. At the present time, the phosphorylation statusof a protein generally is studied by digesting the protein to peptides,and measuring the phosphopeptides by mass spectrometry. Unfortunately,there are problems with sensitivity, specificity, uniform response andmulti-analyte detection. This invention provides a way to overcome theseproblems and can be especially important in bringing some phosphopeptideassays into routine clinical diagnostics where high performance testingis of paramount importance.

A fourth example of an important application of this invention is in theanalysis of phosphoinositides, a class of phosphomonoester-sugars thatare broadly important in medicine and physiology. They are crucialregulators of nuclear functions, cytoskeletal dynamics, cell signalingand membrane trafficking.

A fifth example of an important application of this invention is itsability to discover previously unknown regulatory nucleotides in DNA.Such nucleotides (5-methylcytosine is a known example) would be formedbiosynthetically (in contrast to formation of DNA adducts as aconsequence of damage to DNA).

Materials and Methods for Synthesis'

All chemicals were obtained from commercial suppliers and were usedwithout further purification. NMR were recorded on Varian 300.MALDI-TOF-MS spectra were obtained on an Applied Biosystems Voyager-DESTR. Scintered glass funnels were used for filtrations. Stirring wasdone magnetically. Evaporations were done using a Rotary Evaporator. Allratios of solvents are volume:volume.

Synthesis: General Procedure:

Preparation of Imidazole Reagents (3): General Procedure.

One of compounds 1a-d was dissolved in tetrahydrofuran THF. One molarequivalent each of N-hydroxy succinimide (NHS) and1,3-dicyclohexylcarbodiimide (DCC) were added, respectively. The mixturewas stirred at room temperature (RT) for three hours. Dicyclohexylurea(DCU) was filtered off and the solvents were evaporated. The obtainedN-hydroxy succinimide ester (one of 2a-d) was dried under vacuum anddissolved in acetonitrile. One equivalent of histamine dissolved in 50%acetonitrile/water and three equivalents of triethylamine were added,respectively. The mixture was stirred at room temperature for one hour.Solvents were evaporated and the residue was dried under vacuum andstirred in water. Solid compound (one of 3a-d) was filtered, washed withwater and dried under vacuum. Compound 3e can be prepared similarly.

Synthesis of 3a as a Specific Example.

To 20 ml THF solution of 1.22 g (10 mmol) 1a, 1.18 g (10 mmol) NHS and2.15 g (10 mmol) DCC were added. The mixture was stirred three hours atRT. DCU was filtered and solvents were evaporated. 2a was dried undervacuum. To 10 ml acetonitrile solution of 1.1 g (5 mmol) 2a, 580 mghistamine and 2.1 ml triethylamine in 10 ml acetonitrile/water (50/50,v/v) were added. The mixture was stirred at RT one hour. Solvents wereevaporated, and the residue was further dried under vacuum and thensuspended in 2 ml of water. After 15 min of stirring at RT, 3a wasfiltered, washed with 2 ml of cold water and dried under vacuum. Theyield was 685 mg (63%).

Structural Characterization

2a: ⁻H NMR (ppm) in acetone-d₆: 2.96 (s, 4H), 7.6-7.7 (m, 2H), 7.75-7.84(m, 1H), 8.1-8.2 (m, 2H).

2b: ¹H NMR (ppm) in acetone-d₆: 2.97 (s, 4H), 7.65-7.75 (m, 2H),8.10-8.18 (m, 2H).

2c: ¹H NMR (ppm) in acetone-d₆: 2.97 (s, 4H), 7.84-7.9 (m, 2H), 8.0-8.1(m, 2H).

3a: ¹H NMR (ppm) in methanol-d₄; 2.89 (t, 2H, J=6.9 Hz), 3.61 (t, 2H,J=7.2 Hz), 6.86 (s, 1H), 7.39-7.55 (m, 3H), 7.59 (s, 1H), 7.74-7.80 (m,2H).

3b: ¹H NMR (ppm) in methanol-d₄: 2.88 (t, 2H, J=7.2 Hz), 3.6 (t, 2H,J=7.2 Hz), 6.86 (s, 1H), 7.4-7.5 (m, 2H), 7.59 (s, 1H), 7.74-7.8 (m,2H).

3c: ¹H NMR (ppm) in methanol-d₄: 2.88 (t, 2H, J=7.2 Hz), 3.6 (t, 2H,J=7.2 Hz), 6.86 (s, 1H), 7.55-7.64 (m, 2H), 7.66-7.72 (m, 2H).

3d: ¹H NMR (ppm) in methanol-d₄: 6.86 (s, 1H), 7.4-7.55 (m, 3H), 7.59(s, 1H), 7.65-7.82 (m, 2H). MALDI/MS: (M+H)⁺=220.15.

Materials and Methods for Analysis CCA Matrix

Three mg of α-cyano-4-hydroxycinnamic acid (CCA, 47,687-0, Aldrich,Milwaukee, Wis.), were dissolved in 1.0 ml of acetonitrile:water, 1:1,followed by 1:10 dilution into methanol:water, 1:1, and immediate use.The methanol was from Fisher Scientific, Pittsburgh, Pa.

Settled Propylsulfonic Acid Silica (SPAS)

A 50-ml polypropylene test tube was charged with 20 g of propylsulfonicacid silica (JT Baker, Phillipsburg, N.J.) and 1 M NH₄OH was added up tothe 50-ml mark followed by shaking, settling, pouring off supernatant,and addition of more 1 M NH₄OH (diluted from A669-500, 30%, FisherScientific, Pittsburgh, Pa. 15275) etc. until a pH of about 5 (pH paper)was reached. The supernatant was poured off, and the suspension wastreated similarly with 0.01 M triethylammonium acetate (diluted from 1M, 90359, Fluka, Industriestrasse 25, CH-9471 Buchs SG, Switzerland)until pH 7.0 is attained. The settled product in a total volume of 30 mlwas kept unperturbed at 4° C. and used for at least four months.

Detection Procedure Variations

1. OASIS extraction of DNA adducts. After a sample of DNA was digestedto nucleotides with nuclease P1 and phosphodiesterase I, andultrafiltered (0.5 ml BIOMAX-5 Ultrafree-ML, Millipore, Bedford, Mass.)it was loaded onto an OASIS column (186000383, Waters, Milford, Mass.)that had been washed (gravity flow) once with 1.0 ml methanol and twicewith 0.6 ml of 20 mM triethylammonium acetate, pH 7.0 (TEAA Solution).The sample was loaded followed by washing with 2×0.6 ml then 1 ml ofTEAA Solution, followed by elution with 1.0 ml of 55% methanol in waterinto a 1.5 ml micro centrifuge tube (Fisher Scientific) and evaporationat room temperature in a Speed-Vac.2a. Non-signal imidazole reagent labeling reaction (option for samplederived from step 1). Using an Eppendorf Reference 0.1-2.5 μL Pipettor(Brinkmann, Westbury, N.Y.), 2 μl each of 0.12 M1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC, dissolved in waterand used within 5 min), and 4 μl of a 0.012 M non-signal imidazolereagent in acetonitrile:water, 1:1, were added to the above sample.After aspirating/dispensing the solution six times in order to mix itand also rinse down the sides of the tube, followed by capping, thereaction mixture was allowed to stand in the dark at room temperaturefor 2 h.2b. Non-signal imidazole reagent labeling reaction (option forpre-dissolved sample of a phosphomonoester-containing compound). Usingan Eppendorf Reference 0.1-2.5 μL Pipettor, 1 μl each of anorganophosphate-containing sample in water, 0.12 M EDC (dissolved inwater and used within 5 min), and 4 μl of a 0.012 M non-signal imidazolereagent in acetonitrile:water, 1:1, were combined in a 0.5 mL microcentrifuge tube (Fisher Scientific, Pittsburgh, Pa.). After capping, thereaction mixture was allowed to stand in the dark at room temperaturefor 2 h.3. Ion exchange chromatography. Fifty μl of undisturbed SPAS waspipetted into an UltraMicroSpin Column (The Nest Group, Inc.,Southborough, Mass.), followed by spinning in Micro-Centrifuge Model 59A(Fisher Scientific) at Speed 8 for 10 sec. After similar pipetting andspinning of 2×65 μl of methanol:water, 35:65, the above reaction mixturewas applied onto the center of the ion exchanger and spun into acollection PCR tube (0.2 mL thin-wall PCR tube, 67103-60, Cole Parmer,Vernon Hills, Ill.) followed similarly by 5 μL of methanol:water 35:65.The PCR tube was placed into a 1.5 mL micro centrifuge tube (05-406-17,Fisher Scientific) and its solution was taken to dryness at roomtemperature in a Speed-Vac (−15 min), followed by dissolving in 5 μL ofacetonitrile:methanol:TEAA Solution (6.5:6.5:87).4a. C18 solid phase extraction (first option for step 4). After a ZipTipC18 pipette tip was conditioned by aspirating/dispensing 3×10 μl ofmethanol and 5×10 μl of water with a 10 μl Eppendorf Pipettor, the aboveredissolved sample (in 10 μL of ACN:TEAA solution, 2:98) wasaspirated/dispensed five times (rimming the pipette tip duringdispensing at the bottom of the PCR tube up to the 10 μl level) followedby aspirating/dispensing 5×10 μl of 0.02 M pH 7 triethylammoniumacetate. Elution of the pipette tip onto a MALDI plate was accomplishedas described (http://www.millipore.com/publications.nsf/docs/PS2342ENUS)by using a 10-μl HPLC syringe (80365, Hamilton, Reno, Nev.,) to deliver3 μl each of 5, 10, 20, 30, 50, and 756 methanol in water through theZipTip, with each 0.5 μl of eluent spotted onto a new well of a MALDIplate (384 well plate with hydrophobic plastic surface, 4327695, AppliedBiosystems, Foster City, Calif.), followed by air-drying for 5 min priorto step 5.4b. CapLC column switching (second option for step 4). The samplesolution was injected into a C18-Si trapping column (300 μm id×5 mm,C18, 5 μm, 100A) of a column switching module in a nano-LC system (LCPackings, Dionex Corporation, Sunnyvale Calif.). After washing with TEAASolution:acetonitrile, 99:1 (to retain more polar nucleotides), or 97:3(to remove more of the normal nucleotides) for 3 min at 25 μl/min,switching was done to direct a reverse flow of TEAASolution:acetonitrile:methanol, 90:5:5 (10% organic) onto a micro column(180 μm I.D., 15 cm length, C18-Si, 5 μm, 100 A), and elution was donewith 10 to 35% organic in 0.1×TEAA Solution (2.0 mM) over 7 min, at 2.0μl/min with automated collection of droplets onto a hydrophobic-coatedMALDI plate for 20 sec each using a Probot Micro Fraction Collector (LCPackings, Dionex Corporation), followed by 35% organic for 16 min, 35 to70% organic over 1 min, and 70% organic for 20 min.5. MALDI-TOF analysis. Onto each dried spot from step 4 was applied 0.5μl of CCA matrix. After air-drying for 5 min, the spots on the MALDIplate were analyzed in a MALDI-TOF mass spectrometer (Voyager-DE STR,Applied Biosystems, Foster City, Calif.) in the negative ion mode.Usually the data from 50 but up to 200 laser shots (3 Hz) wereaccumulated to yield a final mass spectrum, at a relative intensitysetting for the nitrogen laser such as 2100. Reflectron mode: grid at68%, source delay time 200 nsec. Linear mode: grid at 94.2%, sourcedelay time 230 nsec.

The structures of some non-signal imidazole reagents that can be used topractice the invention are shown in FIG. 1. A non-signal imidazolereagent consists, e.g., of one or more of these compounds. Compound 1 isN-[2-(1H-imidazol-4-yl)ethyl]benzamide, 2 isN-[2-(1H-imidazol-4-yl)ethyl-d₄]benzamide, 3 is4-chloro-N-[2-(1H-imidazol-4-yl)ethyl]benzamide, 4 is4-bromo-N-[2-(1H-imidazol-4-yl)ethyl]benzamide, and S isN-[2-(1H-imidazol-4-yl)ethyl]benz-d₅-amide. Examples of isotopic duonon-signal imidazole reagents useful in the mass splitting option ofthis invention are as follows: 1+2, 1+5, 2+4, and 3+4.

In FIG. 2 is shown the simultaneous detection of 50 fmol each of ninenucleotides that have been labeled with compound 1. These ninedeoxynucleotides are the four normal deoxynucleotides and the five DNAadducts N²-ethyl-G, 1,N⁶-etheno-A, 8-oxo-G, benzo[a]pyrene-A,benzo[a]pyrene-G, and N²-ethyl-d₄-G, where the G represents dGMP and theA represents dAMP.

5′-Deoxyguanosine monophosphate (5′-dGMP) was successfully detectedstarting with 5 fmol of this compound. In this case option 4b ratherthan 4a was used in the method of the invention. The MALDI-TOF-MSspectrum obtained is shown in FIG. 3, where a signal to noise ratio of100 is seen.

Use of an isotopic duo non-signal imidazole reagent comprising compounds1 and 2 is shown in FIG. 4. In the experiment leading to thisMALDI-TOF-MS spectrum, 100 fmol of an acetylaminofluorene dGMP DNAadduct (AAF-G) was spiked into 300 μg of digested calf thymus DNA afterstep 1. The AAF-G is detected as a pair of peaks at m/z 765.1 and m/z769.1. Also seen in this mass spectrum are peak pairs for three unknownphosphomonoesters in calf thymus DNA that are marked by stars.

DNA from human placenta was subjected to the method of the inventionwith option 4a using an isotopic duo non-signal imidazole reagentconsisting of compounds 1 and 2. This led to the mass spectrum shown inFIG. 5. Along with the four normal deoxynucleotides, unknownphosphomonoesters are detected. The pair of peaks at m/z 571.184 and575.198 correspond to a phosphorimidazolide isotopic duo ofdimethyladenosine monophosphate, suggesting that the DNA sample wascontaminated by RNA.

The detection of ribonucleotides from transfer RNA is shown in FIG. 6.300 μg of tRNA, phenylalanine specific from brewers yeast (R4018, Sigma,St Louis, Mo. 63118) was digested to nucleotides with nuclease P1 andsubjected to steps 2-5 of the method according to the invention usingoption 4a.

Peak Assignments in FIG. 6

-   -   1. 3-methyl-C, 5-methyl-C, 2′-O-methyl-C    -   2. 5-methyl-U, 2′-O-methyl-U, 1-methylpseudo-U,        2′-O-methylpseudo-U    -   3. 1-methyl-A, 2-methyl-A, N⁶-methyl-A, 2′-O-methyl-A    -   4. 1-methyl-G, N²-methyl-G, 7-methyl-G, 2′-O-methyl-G    -   5. N²,N²-dimethyl-G, N²,2′-O-dimethyl-G,        7-aminomethyl-7-deaza-G, 1,2′-O-dimethyl-G    -   6. N⁶-isopentenyl-A    -   7. N⁶-methyl-N⁶-threonylcarbamoyladesosine,        N^(G)-hydroxynorvalylcarbamoyladenosine

Other possible modified nucleotide ions include: pseudo-U (m/2 at 520),dihydro-U (522), 2-thio-C (535), inosine (544), N⁴-acetyl-C (561),5-formyl-2′-O-methyl-C (561), N⁴-acetyl-2′-O-methyl-C (575).

FIG. 7 shows the detection of a phosphopeptide based on labeling with anon-signal imidazole reagent consisting of compound 1. The sequence ofthis pentapeptide is gly-gln-phosphotyr-gly-lys.

5′-Deoxycytidine monophosphate and 5′-deoxyadenosine monophosphate, asderivatives with a non-signal imidazole reagent consisting of compound1, were detected in 150 amol amounts applied to a MALDI target as shownin FIG. 8.

The power of the method is further illustrated by its ability to providethe first direct evidence for N6-methyladenine as a natural component ofmammalian DNA. Many years of testing by many techniques, including manyforms of mass spectrometry, have failed to make this discovery. Incontrast, detection of this modified nucleotide in mammalian DNA is easyby the method of the invention, as shown in FIG. 9. Indeed, in a recentreview of the subject (Ratel D., Ravanat, J.-L., Berger, F., Wion, D.,2006, N6-methyladenine: the other methylated base of DNA, BioEssays 28:309-315), it was stated, “Furthermore, indirect evidence suggests thepresence of m6A in mammal DNA, raising the possibility that this basehas remained undetected due to the low sensitivity of the analyticalmethods used. This highlights the importance of considering m6A as thesixth element of DNA.”

While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

1-9. (canceled)
 10. A non-signal imidazole reagent, said reagent havingthe structure:

wherein either R₁ or R₂ is hydrogen (H) or deuterium (D), B comprisesone to eight carbon atoms, A comprises an aryl group, and at least oneof A or B is substituted with one or more atoms or groups other than Hwhen A is phenyl and B comprises an amide group; and wherein theimidazole reagent has no ionic groups.
 11. The non-signal imidazolereagent of claim 10, wherein B is substituted with four deuterium atoms.12. The non-signal imidazole reagent of claim 10, wherein A issubstituted with a bromine atom (Br), a chlorine atom (Cl), or one ormore deuterium atoms.
 13. The non-signal imidazole reagent of claim 10,wherein A comprises a naphthyl or biphenyl group.
 14. The non-signalimidazole reagent of claim 10, wherein A is substituted with one or morefluorine atoms, alkyl groups or alkoxy groups.
 15. The non-signalimidazole reagent of claim 10, wherein B comprises an amide group or anether group.
 16. The non-signal imidazole reagent of claim 10, wherein Bis —CD₂CD₂- and A is R₃C₆H₄CONH—, wherein R₃ is Br or Cl.
 17. Aphosphorimidazolide anion having the structure:

wherein R₁ comprises an aryl group; R₁ has no ionic groups; and R₂ is anorganic molecule.
 18. The anion of claim 17, wherein R₁ comprises aphenyl group.